CN103524750B - The preparation method of Polyethylene glycol chitosan self-assembled nanometer grain - Google Patents
The preparation method of Polyethylene glycol chitosan self-assembled nanometer grain Download PDFInfo
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Abstract
The preparation method of Polyethylene glycol chitosan self-assembled nanometer grain, belongs to functional materials preparing technical field.The present invention obtains with polyethyleneglycol modified chitosan has chitosan derivatives that is water-soluble and biocompatibility, thus preparation Polyethylene glycol chitosan self-assembled nanometer grain.Advantage of the present invention is: under mild conditions, take TEMEDHCL as thinner, EDC and NHS is activator, by covalent coupling, mPEG-COOH is grafted on CS, prepare with PEG beautify chitosan and a kind of there is Polyethylene glycol chitosan derivative that is water-soluble and good biocompatibility, self-assembly can form nanoparticle in aqueous.
Description
Technical field
The invention belongs to functional materials preparing technical field.
Background technology
Chitosan is the unique alkaline polysaccharide of nature, is the product of chitin N-deacetylation, exists in a large number in shell, shrimp shell crab shell, arthropods, annelid, mollusk.Chitosan has nontoxic, physiologically acceptable, biological degradation and the advantage such as antibacterial, is widely used in the field such as biology, medical science.But due in molecule and the existence of intermolecular powerful hydrogen bond, cause the water insoluble and physiological environment of chitosan, significantly limit its application, therefore, the exploitation with the chitosan derivatives of specified property receives much concern.
In biology, medical field, the preparation method that can be used as the chitosan nano particle of macromolecular drug and the application of bioactive macromolecule carrier mainly contains: covalent cross-linking method, Precipitation method, self-assembly etc.Covalent cross-linking method can affect the release of macromolecular substance; Precipitation method is inhomogeneous reaction, and preparation condition is harsh.Therefore prepare and a kind of there is amphipathic chitose that is water-soluble and good biocompatibility, by molecule in aqueous self-assembly form the nanoparticle of size uniform, having great importance with the embedding and release that realize macromolecular drug and bioactive macromolecule, is the green desirable route of chitosan as pharmaceutical carrier.
Summary of the invention
The object of the invention is to propose a kind of Polyethylene glycol chitosan self-assembled nanometer grain that can overcome above chitosan and chitosan nano preparation method defect.
Molecular structural formula of the present invention is:
Wherein, n=2598, x=300.
The aqueous dispersion of this nanoparticle is stablized, and size uniformity, is suitable for macromolecular drug, the embedding of active macromolecules and release, is expected to become have the bioactive macromolecule of application prospect, the ideal carrier of medicament slow release.
The present invention also proposes the preparation method of above Polyethylene glycol chitosan self-assembled nanometer grain.
Comprise the following steps:
1) mPEG-COOH is prepared: first mPEG is dissolved in dry pyridine, then succinyl oxide is added, isothermal reaction is to terminating, remove pyridine again, gained solid dissolves through toluene, slowly adds normal hexane under magnetic agitation, to product Precipitation, crude product, through dialysis, lyophilize, can obtain the mPEG-COOH that molecular weight is 2000;
2) be that the CS of 500KDa is dissolved in aqueous acetic acid by molecular weight, then dilute CS aqueous acetic acid with the TEMEDHCL that pH is 4.7, obtain CS diluent;
3) by EDC hydrochloride and N-N-N-Hydroxysuccinimide and step 2) the CS diluent made mixes, add mPEG-COOH again, under 35 DEG C of constant temperatures, stir and control pH value of reaction system and maintain 4 ~ 6, the obtained thick product of PEG-CS;
4) PEG-CS crude product is placed in dialysis tubing to dialyse, lyophilize obtains PEG-CS white solid;
5) PEG-CS white solid is scattered in 37 DEG C of distilled water, obtains Polyethylene glycol chitosan self-assembled nanometer grain.
The present invention obtains with polyethyleneglycol modified chitosan has chitosan derivatives that is water-soluble and biocompatibility, thus preparation Polyethylene glycol chitosan self-assembled nanometer grain.
Advantage of the present invention is: under mild conditions, take TEMEDHCL as thinner, EDC and NHS is activator, by covalent coupling, mPEG-COOH is grafted on CS, prepare with PEG beautify chitosan and a kind of there is Polyethylene glycol chitosan derivative that is water-soluble and good biocompatibility, self-assembly can form nanoparticle in aqueous.
In addition, in step 1), the molar ratio of described mPEG and succinyl oxide is 1:1.2, to ensure that mPEG is converted into mPEG-COOH completely; Described constant temperature is 60 DEG C, and reaction 2h, can ensure the homogeneity heated, make to react completely.
Step 2) described in the concentration of aqueous acetic acid be 2%w/v, the feed ratio of described CS and aqueous acetic acid is 1g ︰ 6ml.Can ensure that CS is dissolved in aqueous acetic acid completely.
In step 3), in mPEG-COOH and diluent, the mol ratio of CS is adjustable, to obtain the PEG-CS of certain graft(ing) degree.
In order to ensure that mPEG-COOH activates completely, promote subsequent reactions, in step 3), EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), NHS(N-N-N-Hydroxysuccinimide) and the mixing quality ratio of mPEG-COOH be 16 ~ 77 ︰ 1 ~ 46 ︰ 8 ~ 400.
The molecular weight cut-off of dialysis tubing described in step 4) is 25KDa, dialysis time 4 days, ensures to be dialysed away completely by the small-molecule substance having neither part nor lot in reaction, to obtain pure products.
Accompanying drawing explanation
The infrared spectrogram of Fig. 1 Polyethylene glycol chitosan PEG-CS (PEG:CS=5:1).
The fluorescence spectrum figure of Fig. 2 pyrene in different concns PEG-CS (PEG:CS=5:1).
The I of Fig. 3 pyrene
1/ I
3value is with PEG-CS (PEG:CS=5:1) change in concentration graph of relation.
Fig. 4 Polyethylene glycol chitosan PEG-CS (PEG:CS=5:1) self-assembled nanometer grain TEM photo.
Embodiment
One, embodiment 1: the Polyethylene glycol chitosan self-assembled nanometer grain of preparation PEG:CS=5:1:
1, the synthesis of mPEG-COOH:
Take 5gmPEG and put into round-bottomed flask, dissolve with 25ml dry pyridine, then add 0.3g succinyl oxide, after 60 DEG C of isothermal reaction 2h, remove most pyridine at 45 DEG C with Rotary Evaporators underpressure distillation.Gained solid 30ml toluene dissolves, and magnetic force heating stirrer constantly stirs, slowly adds 40ml normal hexane precipitation agent, separate out to product.Crude product dialysis tubing is dialysed four days, then namely the lyophilize of dialysis gained material is obtained product, can obtain the mPEG-COOH that molecular weight is 2000.
2, Pegylation chitosan PEG-CS(PEG:CS=5:1) preparation:
Take 1gCS(500KDa) be dissolved in 6ml acetic acid (2%, w/v) in, with the TEMEDHCL dilution that 20mlpH is 4.7, join in there-necked flask, then add 0.77mgEDC and 0.46mgNHS, after 30min by mPEG-COOH and CS mol ratio be 5:1 take molecular weight be 2000 20mgmPEG-COOH and molecular weight be the 1gCS(mol ratio of 500KDa be 5:1), join in there-necked flask, water bath with thermostatic control 35 DEG C, stirring reaction 36h, control pH4 ~ 6 in reaction process.
Crude product is placed in dialysis tubing dialysis 4 days, and lyophilize obtains Pegylation chitosan (PEG:CS=5:1) solid.
3, the preparation of Pegylation chitosan PEG-CS (PEG:CS=5:1) self aggregation assemble nanometer grain:
Pegylation chitosan PEG-CS solid is scattered in distilled water 37 DEG C time, obtains Polyethylene glycol chitosan self-assembled nanometer grain.
FT-IR BRUKER, Tensor27 spectrometry.As shown in Figure 1, can be found out by contrast, 1074cm on the IR spectrogram of PEG-CS
-1the relative intensity of left and right ehter bond absorption peak, apparently higher than chitosan, can be observed 1632cm simultaneously
-1, 1522cm
-1the appearance of place's acid amides I bands of a spectrum and acid amides II bands of a spectrum, proves that chitosan and mPEG-COOH there occurs reaction.
Learn that mPEG is successfully grafted to chitosan by Fig. 1, under the effect of EDC, there is acylation in the carboxyl on mPEG-COOH and the amino on chitosan, form amido linkage, mPEG is grafted to chitosan, and PEG-CS molecular structure as shown in Figure 2.
Take pyrene as fluorescent probe, study PEG-CS nano particle Assembling Behavior in aqueous with fluorescence spectrum (F4500 type fluorescence meter).As shown in Figure 2,3, in the PEG-CS solution of lower concentration, the I of pyrene
1/ I
3value is close to the I of pyrene in pure water
1/ I
3value 1.72, when PEG-CS concentration is 0.5 ~ 1.4mg/mL, I
1/ I
3value significantly decreases, when PEG-CS concentration is greater than 1.4mg/mL, and I
1/ I
3be worth constant in about 1.20, although be less than the value in pure water, interface is still very hydrophilic.Repeat to test after PEG-CS being placed 7 days and obtain identical result, show that Polyethylene glycol chitosan self-assembled nanometer grain has good stability.
Fig. 4 is the photo after the dyeing of PEG-CS self-assembled nanometer grain phospho-wolframic acid, Tecnai-12 type transmission electron microscope observation.PEG-CS self-assembled nanometer grain is regular spherical as can be seen from Figure, and particle diameter is 35 ± 5nm about, has very bright and clean surface and good regularity.
Prepared Pegylation chitosan self-assembled nanometer grain can be used as pharmaceutical carrier, increases the solubleness of medicine, metabolic stability and circulation time in vivo.As Pegylation chitosan nano prepared herein can successful load insulin, encapsulation rate can reach more than 85%.
Molecular structural formula is:
Wherein, n=2598, x=300.
Two, embodiment 2: the Polyethylene glycol chitosan self-assembled nanometer grain of preparation mPEG:CS=1:10:
1, the synthesis of mPEG-COOH:
Take 5gmPEG and put into round-bottomed flask, dissolve with 25ml dry pyridine, then add 0.3g succinyl oxide, after 60 DEG C of isothermal reaction 2h, remove most pyridine at 45 DEG C with Rotary Evaporators underpressure distillation.Gained solid 30ml toluene dissolves, and magnetic force heating stirrer constantly stirs, slowly adds 40ml normal hexane precipitation agent, separate out to product.Crude product dialysis tubing is dialysed four days, then namely the lyophilize of dialysis gained material is obtained product, can obtain the mPEG-COOH that molecular weight is 2000.
2, the preparation of Pegylation chitosan PEG-CS (PEG:CS=1:10):
Take 1gCS (500KDa) and be dissolved in 6ml acetic acid (2%, w/v) in, with 20mlTEMEDHCL(pH4.7) dilution, join in there-necked flask, then EDC (0.016mg) and NHS (0.01mg) is added, after 30min in the ratio of mPEG:CS=1:10 take 0.4mg molecular weight be 2000 mPEG-COOH and 1g molecular weight be that the CS(molar mass of 500KDa is than for 1:10), join in there-necked flask, water bath with thermostatic control 35 DEG C, stirring reaction 36h, control pH4-6 in reaction process.
Crude product is placed in dialysis tubing dialysis 4 days, and lyophilize obtains Pegylation chitosan (PEG:CS=1:10) solid.
3, the preparation of Pegylation chitosan PEG-CS (PEG:CS=1:10) self aggregation assemble nanometer particle:
Pegylation chitosan PEG-CS (PEG:CS=1:10) solid is scattered in distilled water 37 DEG C time, obtains Polyethylene glycol chitosan self-assembling nanoparticles.
Molecular structural formula is:
Wherein, n=2598, x=300.
Claims (5)
1. the preparation method of Polyethylene glycol chitosan self-assembled nanometer grain, molecular structural formula is:
Wherein, n=2598, x=300, the preparation method of described Polyethylene glycol chitosan self-assembled nanometer grain comprises the following steps:
1) prepare mPEG-COOH: first mPEG is dissolved in dry pyridine, then add succinyl oxide, isothermal reaction is to terminating, removing pyridine, gained solid dissolves through toluene, slowly adds normal hexane under magnetic agitation, to product Precipitation, crude product obtains mPEG-COOH through dialysis, lyophilize;
2) be that the CS of 500KDa is dissolved in aqueous acetic acid by molecular weight, then dilute CS aqueous acetic acid with the TEMEDHCL that pH is 4.7, obtain CS diluent;
3) by EDC hydrochloride and N-N-N-Hydroxysuccinimide and step 2) the CS diluent made mixes, add mPEG-COOH again, under 35 DEG C of constant temperatures, stir and control pH value of reaction system and maintain 4 ~ 6, the obtained thick product of PEG-CS;
4) PEG-CS crude product is placed in dialysis tubing to dialyse, lyophilize obtains PEG-CS white solid;
5) PEG-CS white solid is scattered in 37 DEG C of distilled water, obtains Polyethylene glycol chitosan self-assembled nanometer grain.
2. preparation method according to claim 1, is characterized in that in step 1), and the molar ratio of described mPEG and succinyl oxide is 1:1.2, and described temperature of reaction is 60 DEG C of constant temperature, reaction 2h.
3. preparation method according to claim 1, is characterized in that step 2) described in the concentration of aqueous acetic acid be 2%w/v, the feed ratio of described CS and aqueous acetic acid is 1g ︰ 6ml.
4. preparation method according to claim 1, is characterized in that the mixing quality ratio of the hydrochloride of EDC described in step 3), N-N-N-Hydroxysuccinimide and mPEG-COOH is 16 ~ 77 ︰ 1 ~ 46 ︰ 8 ~ 400.
5. preparation method according to claim 1, is characterized in that the molecular weight cut-off of dialysis tubing described in step 4) is 25KDa, dialysis time 4 days.
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| CN104491844B (en) * | 2014-12-02 | 2017-02-22 | 扬州大学 | Production method of insulin oral sustained-release preparation |
| RU2642786C2 (en) * | 2015-01-30 | 2018-01-26 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) | Liposomal suspensions stabiliser |
| CN105640922B (en) * | 2016-01-25 | 2018-08-17 | 扬州大学 | The preparation method of the positively charged arsenic trioxide preparation with Liver targeting effect |
| CN106750336A (en) * | 2016-12-08 | 2017-05-31 | 福州大学 | A kind of preparation method of glucan co polyethylene glycol nanoparticles |
| CN109485747B (en) * | 2017-09-12 | 2020-12-11 | 中国科学院理化技术研究所 | Water-soluble chitosan antibacterial derivative and preparation method thereof |
| CN111484568B (en) * | 2019-01-25 | 2021-12-14 | 中国科学院理化技术研究所 | Chitosan-antibacterial polypeptide graft polymer and preparation method and application thereof |
| CN109912850B (en) * | 2019-03-11 | 2021-03-02 | 同济大学 | Self-healing hydrogel entrapping exosomes and preparation method and application thereof |
| CN110628030A (en) * | 2019-05-28 | 2019-12-31 | 济南大学 | Alfa-linolenic acid modified monomethoxy polyethylene glycol-chitosan oligosaccharide and preparation thereof |
| CN110384684B (en) * | 2019-08-26 | 2021-04-06 | 安徽农业大学 | Monocarboxyl chitosan/alkannin composite nano-particles and preparation method thereof |
| CN112300408B (en) * | 2020-10-26 | 2022-10-04 | 广州大学 | Flexible high-tensile temperature-sensitive and humidity-sensitive hydrogel and preparation method and application thereof |
| CN112592416A (en) * | 2020-12-31 | 2021-04-02 | 重庆科技学院 | Preparation method and application of 3- (9-anthracene) acrolein/ethylene glycol chitosan |
| CN114984238B (en) * | 2022-06-25 | 2024-01-30 | 中国海洋大学 | Multifunctional amphiphilic self-assembled nano-carrier based on chitosan and preparation method thereof |
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